Production of Cellulose Nanocrystals From Raw Wood Via Hydrothermal Treatment

ABSTRACT

A method comprises subjecting raw wood comprising cellulose, lignin and hemicellulose to a heat treatment at a crystallization temperature in the range of from about 150° C. to about 250° C. for a period of time sufficient to induce crystallization of cellulose in the raw wood, wherein the crystallinity of the processed raw wood as measured after delignification using 380 Raman is at least 5% greater than the crystallinity of the raw wood as measured after delignification and prior to processing using 380 Raman. The method can further comprise extracting CNCs from the processed raw wood via an acid hydrolysis procedure.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Patent Application No. 62/307,271 that was filed Mar. 11, 2016, the entire contents of which are hereby incorporated by reference.

REFERENCE TO GOVERNMENT RIGHTS

This invention was made with government support. The government has certain rights in the invention.

BACKGROUND

The structure of wood cell wall cellulose in its native state remains poorly understood, limiting the progress of research and development in numerous areas, including plant science, biofuels, and nanocellulose based materials. It has generally been believed that cellulose in cell wall microfibrils has both crystalline and non-crystalline regions. On this basis, cellulose nanocrystals (CNCs) for use in a variety of applications have been prepared from chemically pulped wood sources.

SUMMARY

Methods for forming crystalline cellulose in raw wood are provided. Also provided are methods for extracting cellulose nanocrystals (CNCs) from the processed raw wood as well as the extracted CNCs.

In one aspect, methods for forming crystalline cellulose in raw wood are provided. In an embodiment, the method comprises subjecting raw wood comprising cellulose, lignin and hemicellulose to a heat treatment at a crystallization temperature in the range of from about 150° C. to about 250° C. for a period of time sufficient to induce crystallization of cellulose in the raw wood, wherein the crystallinity of the processed raw wood as measured after delignification using 380 Raman is at least 5% greater than the crystallinity of the raw wood as measured after delignification and prior to processing using 380 Raman.

In another aspect, methods of producing cellulose nanocrystals (CNCs) from raw wood are provided. In an embodiment, the method comprises subjecting raw wood comprising cellulose, lignin and hemicellulose to a heat treatment at a crystallization temperature in the range of from about 150° C. to about 250° C. for a period of time sufficient to induce crystallization of cellulose in the raw wood, wherein the crystallinity of the processed raw wood as measured after delignification using 380 Raman is at least 5% greater than the crystallinity of the raw wood as measured after delignification and prior to processing using 380 Raman; and extracting CNCs from the processed raw wood via an acid hydrolysis procedure.

In another aspect, cellulose nanocrystals extracted from the processed raw wood are provided. In an embodiment, cellulose nanocrystals comprise lignin and have a Klason lignin content of about 20% or greater.

Other principal features and advantages of the invention will become apparent to those skilled in the art upon review of the following drawings, the detailed description, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative embodiments will hereafter be described with reference to the accompanying drawings.

FIG. 1 shows the overlaid Raman spectra for lignin-free wood-CNCs and lignin-free pulp-CNCs.

FIG. 2 shows a TEM image of lignin-free wood-CNCs at 46 K magnification.

FIG. 3 shows a TEM image of lignin-free pulp-CNCs at 46 K magnification.

DETAILED DESCRIPTION

Methods for forming crystalline cellulose in raw wood are provided. Also provided are methods for isolating cellulose nanocrystals (CNCs) from the processed raw wood as well as the isolated CNCs. The methods are based, in part, upon the discovery that the crystallinity of cellulose in raw wood is significantly less than had been conventionally thought. However, it has been found that crystallization of cellulose in raw wood can be induced by an appropriate heat treatment. Without wishing to be bound to a particular theory, it is thought that the appropriate heat treatment causes transformation of CH₂OH groups of cellulose from the gt conformation to the tg conformation. Only the tg conformation can participate in intra-plane, hydrogen bond formation. Within the ordered phase of cellulose, transformation of gt retains an additional degree of freedom to transition gt to tg and vice versa. The heat treatment completely converts gt to tg and eliminates the degree of freedom for reversibility. It has further been found that CNCs extracted from raw wood processed according to the present crystallization methods are capable of exhibiting improved properties as compared to CNCs extracted from pulped wood (raw wood which has been subjected to pulping treatments as further described below). For example, CNCs extracted from raw wood processed using at least some of embodiments of the present crystallization methods exhibit one or more of the following improved properties as compared to CNCs extracted from pulped wood: higher crystallinity, less water accessibility, greater lengths, and greater uniformity in widths.

In one aspect, a method for forming crystalline cellulose in raw wood is provided. In embodiments, the method comprises subjecting raw wood to a heat treatment at a crystallization temperature for a period of time sufficient to induce crystallization of cellulose in the raw wood. The raw wood comprises cellulose, lignin and hemicellulose. Multiple cellulose molecular chains associate to form individual cellulose fibers. In raw wood, cellulose fibers are bound together in a matrix of lignin and hemicellulose. The phrase “raw wood” is distinguished from raw wood which has been subjected to a pulping treatment to induce separation of cellulose fibers to convert the raw wood to a pulp. Phrases such as “pulped wood” and the like are used to refer to raw wood subjected to such pulping treatments. These pulping treatments include chemical pulping treatments (and their variations, e.g., thermochemical pulping treatments) which involve the use of chemicals to dissolve the lignin in the raw wood as well as mechanical pulping treatments (and their variations, e.g., thermomechanical pulping treatments) which involve the use of mechanical force to induce cellulose fiber separation, but do not necessarily remove the lignin.

The raw wood may be derived from hardwood (non-coniferous) sources as well as softwood (coniferous) sources.

The raw wood may be provided in various physical forms, e.g., wood meal, wood chips, milled wood, wood shavings, etc. Wood meal may be characterized as particles of raw wood having a predetermined size or size distribution. By way of illustration, 40-mesh wood meal (used in some of the Examples below), refers to particles of raw wood which have been passed through a 0.4 mm mesh screen. Thus, the particles of raw wood in 40-mesh wood meal have a largest dimension of less than about 0.4 mm. In addition, the raw wood may be subjected to extractives removal prior to the heat treatment. By way of illustration, multiple soaks in organic solvent (e.g., acetone):water mixtures at room temperature followed by air drying may be used to remove extractives in the raw wood prior to use as described in the Examples below.

The raw wood may be characterized by its lignin content. The lignin content may be a Klason lignin content reported as a percentage and determined as described in the Examples, below. Thus, the disclosed lignin contents refer to lignin content determined using the method described in the Examples, below. The raw wood may have a Klason lignin content in the range of from about 17% to about 33%.

The raw wood may be provided as a wood blend comprising the raw wood, water and optionally, additives. Illustrative additives include various salts and buffers. Salts may be selected to modify the hydrogen bonding in cellulose. Buffers may be selected to achieve a desired pH. Illustrative salts and buffers are provided in Example 2, below. However, by contrast to chemical pulping treatments, additives used in the wood blend do not include chemicals capable of dissolving the lignin in the raw wood. Such chemicals include sulfates, sulfites and/or sulfides (e.g., sulfide salts, bisulfate salts, sulfurous acid, sulfuric acid, etc.). In embodiments, the wood blend consists essentially of, or consists of, the raw wood and water.

As noted above, the heat treatment in the present crystallization methods involves a crystallization temperature which is an elevated temperature, e.g., greater than 70° C. Comparative Example 1, below, shows that without such a heat treatment, cellulose nanocrystals (CNCs) cannot be extracted from raw wood. The crystallization temperature is generally in the range of from about 150° C. to about 250° C. Within this range, the heating time may be adjusted to provide a desired crystallinity, e.g., maximum crystallinity. Example 2, below, shows that temperatures less than about 150° C. provide reduced crystallinity. However, the temperature generally does not exceed 250° C. in order to prevent degrading the cellulose. In embodiments, the crystallization temperature is in the range of from about 160° C. to about 240° C., from about 170° C. to about 230° C., from about 175° C. to about 230° C., or from about 180° C. to about 230° C. The time which the raw wood is exposed to the crystallization temperature may be in the range of from about 60 minutes to about 120 minutes, about 70 minutes to about 110 minutes, or about 80 minutes to about 100 minutes.

Although the wood blend may be mixed, stirred, or the like during the heat treatment, the heat treatment does not involve directly applying mechanical force to the raw wood. As such, the heat treatment may be characterized as being carried out without subjecting the raw wood to substantial mechanical force. This is by contrast to mechanical pulping treatments.

As noted above, the heat treatment induces the crystallization of the cellulose in the raw wood. The crystallinity of the raw wood processed by the present crystallization methods may be characterized using Raman spectroscopy as described in Example 1, below. The Raman spectroscopic method described in Example 1, below, may be referred to as “380 Raman.” Such processed raw wood may be subjected to a delignification procedure prior to determining crystallinity. A suitable delignification procedure is described in Example 2, below. Crystallinity as determined using 380 Raman is reported as a percentage, wherein a higher percentage corresponds to a greater degree of crystallinity, i.e., a greater degree of order between cellulose molecular chains. In embodiments, the processed raw wood (after delignification) is characterized by a crystallinity of at least 50%, as measured by 380 Raman. This includes embodiments in which the processed raw wood (after delignification) is characterized by a crystallinity of at least 54%, at least 60%, at least 65%, or at least 70% as measured by 380 Raman.

The crystallinity of raw wood processed by the present crystallization methods may be characterized by its accessibility to water. Water accessibility refers to the percentage of CH₂OH groups on cellulose which are accessible to water as compared to completely amorphous cellulose in which 100% of the CH₂OH groups are assumed to be accessible to water. Water accessibility is another indicator of crystallinity as crystalline cellulose is less accessible to water due to the greater degree of order between neighboring chains. Water accessibility may be determined via OH to OD exchange coupled with Raman spectroscopy as described in Example 1, below. The OH/OD exchange coupled with Raman spectroscopy technique described in Example 1, below, may be referred to as “1380 Raman.” Again, the processed raw wood may be subjected to a delignification procedure prior to determining its accessibility to water. In embodiments, the processed raw wood (after delignification) is characterized by an accessibility to water of no more than 25% as measured using 1380 Raman. This includes embodiments in which the processed raw wood (after delignification) is characterized by an accessibility to water of no more than 24%, no more than 22%, or no more than 20% using 1380 Raman.

Regardless of the technique used to measure crystallinity (i.e., either 380 Raman or 1380 Raman), the crystallization methods are capable of increasing the crystallinity of the processed raw wood. The magnitude of the increase can be determined by comparing the crystallinity of the processed raw wood (after delignification) to the crystallinity of the raw wood (after delignification) prior to processing. In embodiments, the increase is at least about 5%, at least about 7%, at least about 9%, at least about 11% or at least about 13% as measured using 380 Raman.

The raw wood processed by the present crystallization methods may be characterized by its lignin content. The lignin content is provided as a Klason lignin content reported as a percentage and determined as described above. The processed raw wood may have a Klason lignin content in the range of from about 20% to about 50%.

The present crystallization methods may be carried out in various reactor systems, e.g., a Parr reactor or a Berghof reactor.

In another aspect, a method of producing cellulose nanocrystals (CNCs) from raw wood is provided. In embodiments, the method comprises extracting CNCs from the raw wood processed by the present crystallization methods described above. Various techniques may be used to extract the CNCs, including acid hydrolysis. Acid hydrolysis involves exposing the processed raw wood to an acid (e.g., aqueous acid solution) at a hydrolysis temperature and for a time sufficient to hydrolyze cellulose molecules present in non-crystalline domains. Illustrative acids, temperatures and times are described in the Examples, below. The processed raw wood may be used as is or washed with room temperature (i.e., 20 to 25° C.) water and dried prior to the acid hydrolysis. In either case, the extracted CNCs will include lignin (since the raw wood includes lignin) and may be referred to as “lignin-containing wood-CNCs.” Lignin-containing wood-CNCs are advantageous since they are hydrophobic and easily dispersible in various matrices.

Alternatively, the processed raw wood may be subjected to a delignification procedure (see Example 2) prior to the acid hydrolysis. In that case, the extracted CNCs will be substantially free of lignin and may be referred to as “lignin-free wood-CNCs.” The term “substantially” is used to indicate use of a delignification procedure to remove the lignin although a small amount of lignin (e.g., modified lignin) may still be present.

The yield of cellulose nanocrystals (CNCs) extracted may be determined using the equation Yield=(weight of the freeze-dried CNCs extracted/weight of the oven dried starting wood)*100. (See Example 1.) The yield of lignin-containing wood-CNCs may be in the range of from about 4% to about 20%.

The cellulose nanocrystals (CNCs) are elongated, rod-like nanoparticles comprising multiple cellulose molecular chains. The surface of the CNCs have been functionalized with sulfate esters during the digestion with sulfuric acid. The CNCs may be characterized by an average length and an average width. By “average” it is meant an average value as determined from measuring the lengths/widths of a population of CNCs. The width dimension may be measured from atomic force microscope (AFM) or transmission electron microscope (TEM) images as described in Example 2, below. The length dimension may be measured from TEM images as described in Example 2, below. In embodiments, the average length of the CNCs as determined from TEM images is in the range of from about 30 nm to about 330 nm or from about 50 nm to about 300 nm. In embodiments, the average width of the CNCs as determined from AFM images is in the range of from about 7 nm to about 8.5 nm. In embodiments, the average width of the CNCs as determined from TEM images is in the range of from about 4 nm to about 20 nm or from about 5 to about 16 nm. The CNCs may also be characterized by a width distribution, i.e., the difference between the minimum and maximum widths measured from a population of CNCs using AFM or TEM images. In embodiments, the width distribution of the CNCs as determined from AFM images is less than about 2 nm, less than about 1.5 nm, or less than about 1 nm. As compared to lignin-free pulp-CNCs, the wood-CNCs (both lignin-containing and lignin-free) are more uniform and have narrower widths.

The cellulose nanocrystals (CNCs) may be characterized by their crystallinity, determined as described above with respect to the raw wood processed using the present crystallinity methods. The crystallinity values of the CNCs may include the ranges described above with respect to the processed raw wood. Similarly, the CNCs may be characterized by their accessibility to water, determined as described above with respect to the processed raw wood. The water accessibility values of the CNCs may be within the ranges described above with respect to the processed raw wood. As compared to lignin-free pulp-CNCs, the lignin-free wood-CNCs are more crystalline and are less accessible to water. This is advantageous since such CNCs are less likely to suffer from moisture induced property changes.

As described above, the heat treatment to induce crystallization of cellulose is applied directly to raw wood as opposed to pulped wood. However, the heat treatment may also be applied to pulped wood as the starting material, e.g., mechanically pulped wood. Therefore, in embodiments, mechanically pulped wood can be subjected to a heat treatment at a crystallization temperature in any of the disclosed ranges for a period of time sufficient to induce crystallization of cellulose in the mechanically pulped wood. The crystallinity values of the processed mechanically pulped wood may be within the ranges described above. Cellulose nanocrystals may be extracted from the processed mechanically pulped wood as described above with respect to processed raw wood. Optionally, the processed mechanically pulped wood may be subjected to a delignification procedure prior to CNC extraction.

EXAMPLES Comparative Example 1: Extraction of Cellulose Nanocrystals (CNCs) from Raw Wood without Heat Treatment

Air-dried loblolly pine wood (31.5 g of 40-mesh meal) was added to a three-neck, round-bottom flask equipped with a mechanical mixer, addition funnel, and barbed stopcock. The flask was flushed with N₂, evacuated (3 mm Hg), filled with N₂, and evacuated (3 mm Hg). Next, 250 ml 64% H₂SO₄ (room temp) was added over 1 min. The wood mass quickly turned very dark, initially appearing as a paste but the viscosity dropped in about 15 min. The acid hydrolysis reaction was carried out for 90 min at 45° C. The contents were diluted to 4300 mL and the solids containing portion settled to approximately 1 L. The solution was decanted then diluted to about 2.5 L. The suspension was neutralized with NaOH solution (800 ml at 5 wt %) followed by dilution to 4300 mL. The suspension was allowed to settle overnight. Solids settled to approximately 1 L. The solution was decanted and the suspended solids dialyzed against RO (reverse osmosis) water for 1 week with daily flipping of the dialysis tubes to periodically mix the significant amount of dark solids that settle to the bottom relatively quickly. The dialyzed suspension (1.4 L) was treated with an ultrasonic probe (20k Hz) for 12 min (100,000 J). The suspension was then centrifuged for 15 min at 10,000×g and decanted. The solids were washed once by suspending again to 1 L and centrifuging at 10,000×g for 15 min. This decant was combined with the previous one and the total concentrated to about 20 mL using a rotovap and then dried under vacuum over P₂O₅ to yield 0.58 g solids. The sugar and lignin analysis is shown in Table 1 below in which this sample is referred to as SA15. Although this sample has 30% glucan, the glucan yield is only 1.4% glucan-yield based on 40% cellulose in the wood. The dark solids retained by the centrifugation were air-dried to 11.7 g. The sugar and lignin analysis is shown in Table 1 in which this sample is referred to as sample SA16. The glucan yield is ˜12.8% based on 40% cellulose in wood.

The amount of Klason lignin was determined by using the TAPPI test method as described in “Acid insoluble lignin in wood and pulp; official test method T-222 (Om),” TAPPI, Atlanta, Ga., 1983, which is hereby incorporated by reference in its entirety. The amounts of carbohydrates were determined according to Davis, M. W., “A rapid method for compositional carbohydrate analysis of lignocellulosics by high pH anion-exchange chromatography with pulse amperometric detection (HPAE/PAD),” J. Wood Chem. Technol. 1998, 18:235-252, which is hereby incorporated by reference in its entirety.

TABLE 1 Chemical Analysis of Hydrolysis Fractions of Raw Wood Without Heat Treatment. Klason lignin Hemicellulose^(a) (%) Arabinan (%) Galactan (%) Glucan (%) Xylan (%) Mannan (%) (%) SA15^(b) (fraction 38.1 0.1 0.2 30.0 0.7 1.0 2.0 with CNCs, if present) SA16^(c) 75.8 0.1 0.2 13.8 0.6 0.6 1.5 (membrane filtered fraction) ^(a)Hemicellulose is sum of xylan, mannan, Arabian, and galactan. Because pine has glucomannan, part of the glucose is hemicelluloses as well. ^(b)This fraction had higher glucan content compared to SA16, indicating no CNCs. ^(c)High lignin fraction that passed through the membrane filter.

In SA15 (the fraction which would include any CNCs if present) the glucan yield of 1.4% (based on wood mass) was considered to be negligible and it was concluded that the loblolly pine hardly produced any CNCs. In addition, the bottle containing the suspension of the fraction which would include any CNCs if present was mostly transparent, further confirming the lack of CNCs. This example was repeated with delignified wood meal, but the results were similar, indicating that the presence of lignin does not affect the generation of CNCs. These results have also been described in Agarwal, U. P., et al., “Probing Crystallinity of Never-Dried Wood Cellulose with Raman Spectroscopy.” Cellulose 2016, 23 (1), 125-144, which is hereby incorporated by reference in its entirety.

Comparative Example 2: Extraction of Cellulose Nanocrystals (CNCs) from Chemically Pulped Wood

Torn pieces of bleached softwood kraft pulp dry lap (7.9 g, 5% moisture) were added to a three-neck, round-bottom flask equipped with a mechanical mixer, addition funnel and barbed stopcock. The flask was flushed with N₂, evacuated (3 mm Hg), filled with N₂, and evacuated (3 mm Hg). Next, 80 ml 64% H₂SO₄ (room temp) was added in 1 min. The dry lap swells as it absorbs the acid and initially becomes a paste as stirred but the viscosity drops in about 15 min. The acid hydrolysis reaction was carried out for 60 min at 45° C. As the reaction proceeds, the reaction turns light yellow, but gradually darkens to a light brown as sugars degrade during the reaction. The contents were diluted to 2500 mL. Sodium chlorite (0.1 g) was added to the acidic CNC suspension, creating ClO₂ that bleaches the degraded sugars. After 30 minutes, the suspension is neutralized with IL of 6% NaOH solution. Everything is diluted to 4.3 L and the suspension was allowed to settle overnight. Solids settled to approximately 0.5 L. The solution was decanted and the suspended solids dialyzed against RO (reverse osmosis) water for one week with daily flipping initially until solids no longer settled to the bottom of the dialysis tube. The dialyzed suspension (˜1 L) was treated with ultrasonic probe (20 k Hz) for 2 minutes (25,000 Joules). The suspension was then centrifuged for 15 minutes at 10,000×g and decanted; only a trace of solids were visible at the corners of the bottles. This colloidal CNC suspension was concentrated to about 50 mL using a rotovap. A small amount of t-butanol (about 10% by volume) was added and the mixture frozen in about 15 minutes by placing it in an ice-salt bath (about −20° C.) then freeze-dried to yield 1.95 g, a ˜25% yield on starting pulp. That is, the yield for lignin-free pulp-CNCs based on starting pulp was determined from the following equation: ((freeze-dried solids weight 1.95 g)/starting pulp weight of 7.9 g))*100). The yield for lignin-free pulp-CNCs based on wood was then ˜13%, assuming on average 50% yield of the starting pulp from wood (this pulp yield from is generally accepted). Characteristics of the lignin-free pulp-CNCs are shown in Table 3, below, and are further described with respect to the next Example, Example 1.

Example 1: Extraction of Cellulose Nanocrystals (CNC) from Raw Wood With Heat Treatment

Poplar wood meal was produced by Wiley milling chips to pass a screen with 1 mm holes. Moist poplar wood meal (150 g, 68.9% solids, 103.4 g O.D. wood (E, in Table 2)) was soaked with about 1 L of acetone:water (9:1) overnight at room temperature. The wood was filtered and soaked in another liter of acetone:water for four hours at room temperature. The wood was filtered and soaked a third time in acetone:water for four hours at room temperature. The wood meal was allowed to air-dry to constant weight (100.9 g, 97.7% yield (D, in Table 2). Extracted wood meal (100 g) was mixed with 900 mL of 0.1 M phosphate buffer (Na₂HPO₄:NaH₂PO₄ molar ratio 5:1); the initial pH was 7.5. This was heated (ramp 45 minutes) in a 2 L horizontal Parr reactor with mixing to 170° C. and held at this temperature for 45 minutes. After cooling quickly, the final pH was measured at 5.2. The wood meal was filtered, washed well with deionized (DI) water and allowed to air dry (87.6 g, 87.6% yield from extracted wood (C, in Table 2)). Treated wood meal (15 g) was mixed with 120 mL 64 wt % sulfuric acid under vacuum, then placed under a nitrogen atmosphere and stirred in a 45° C. water bath for 90 minutes. Then the acid hydrolysis reaction was quenched by dilution to about 2 L. This suspension was neutralized using a 5 wt % NaOH solution. The suspension was diluted to 4.5 L and allowed to settle overnight. About 3.5 L of clear brown supernatant was decanted. The remaining suspension was transferred to a dialysis tube and dialyzed against laboratory DI water for one week. The dialyzed suspension (900 mL) was treated with ultrasonic probe (20k Hz) for 12 minutes (100,000 Joules). The suspension was then centrifuged for 15 minutes at 10,000×g and decanted. The solids were washed once by suspending again to 800 mL and centrifuging at 10,000×g for 15 min. This decant was combined with the previous one and the total concentrated to about 42.9 g using a rotovap; a fraction was freeze dried to determine the solids content at 4.50% (1.93 g total solids, 11.3% yield from oven dried starting wood meal (A, in Table 2)). The lignin-containing wood-CNC yield was determined from the following equation: ((weight of solids from decant)/(weight of oven dried starting wood meal))*100. The solids from the centrifuge were also freeze dried (3.23 g, 18.8% yield oven dried starting wood meal (B, in Table 2)).

Chemical analysis of the various fractions A-E was conducted as described in Comparative Example 1, above. The results are shown in Table 2.

TABLE 2 Chemical Analysis of Fractions A-E.^(a) Total Klason Lignin Ash Arabinan Galactan Carbohydrate Total Yield Fraction lignin (%) (%) (%) (%) Glucan (%) Xylan (%) Mannan (%) (%) (%)^(b) A 30.6 −1.0 ND ND 56.44 1.01 ND 57.45 88.1 B 83.9 −0.3 ND ND 5.46 0.55 ND 6.01 89.91 C 22.6 −0.3 ND 0.53 51.34 10.58 2.65 65.10 87.7 D 21.2 −0.5 ND 0.72 47.73 13.02 2.89 64.36 86.6 E 21.7 −0.2 ND 0.64 46.37 12.24 2.58 61.82 83.5 ^(a)ND is not detected. ^(b)Sum of Klason lignin and total carbohydrate.

As noted above, the extracted CNCs include lignin (lignin-containing wood-CNCs) because the heat treated raw wood includes lignin. Lignin-free wood-CNCs were obtained using a standard acid chlorite delignification procedure prior to the extraction of the CNCs as described in Example 2, below.

The lignin-free CNCs extracted from wood pulp (lignin-free pulp-CNCs) obtained in Comparative Example 2, the lignin CNCs and lignin-free CNCs extracted from raw wood (lignin-containing wood-CNCs and lignin-free wood-CNCs) obtained in Example 1 were analyzed via Raman spectroscopy, X-ray diffraction (XRD), transmission electron spectroscopy (TEM), atomic force microscopy (AFM), and inductively coupled plasma atomic emission spectroscopy (ICP-AES). These techniques were used to estimate the following properties of the CNCs: crystallinity (380 Raman and XRD), accessibility to water (1380 Raman), average particle width (AFM and TEM), particle uniformity (AFM and TEM), and surface sulfonation (ICP-AES). The data are shown in Table 3, below. FIG. 1 shows the overlaid Raman spectra for lignin-free wood-CNCs and lignin-free pulp-CNCs. FIG. 2 shows a TEM image of lignin-free wood-CNCs at 46 K magnification. FIG. 3 shows a TEM image of lignin-free pulp-CNCs at 46 K magnification.

Regarding the Raman analysis (crystallinity and accessibility to water), samples were analyzed with a Bruker MultiRam spectrometer (Bruker Instruments Inc., Billerica, Mass.). This Raman system is equipped with a 1064-nm 1000-mW continuous wave (CW) diode pumped Nd:YAG laser. Samples were prepared in two ways for analysis-either by making a pellet in a pellet press or by sampling in shortened NMR glass tubes. Some samples were sampled in both ways. Numerous samples were analyzed in presence of H₂O and D₂O in addition to analyzing them in the dry state in the tube. This was done to study the effect of OH to OD exchange as a measure of the accessibility of the cellulose to water. For Raman crystallinity estimation purposes, spectra were obtained from pellets of 0.1 g. (See Agarwal, U. P., et al., “Cellulose I crystallinity determination using FT-Raman spectroscopy: univariate and multivariate methods,” Cellulose 2010, 17: 721-733 (“Agarwal 2010”) and Agarwal, U. P., et al., “Estimation of Cellulose Crystallinity of Lignocelluloses Using Near-IR FT-Raman Spectroscopy and Comparison of the Raman and Segal-WAXS Methods,” J. Agric. Food Chem. 2013, 61: 103-113 (“Agarwal 2013”), each of which is hereby incorporated by reference in its entirety. The laser power used for sample excitation was 900 mW but in cases where significant fluorescence was encountered or the possibility of thermal degradation existed, the power was reduced to 600 mW. Depending upon the S/N desired and the concentration of the sample (in H₂O or D₂O) anywhere from 1024 to 16,384 scans were co-added. Some samples (e.g., CNCs in H₂O or D₂O) were quite dilute and to get better S/N ratio 16,384 scans were obtained. OH to OD exchange was performed by removing excess water, putting the sample in D₂O (99.9% deuterated), then centrifuging (4000×g) the sample in NMR tube, and removing excess D₂O. Thereafter, this process was repeated in the sampling tube itself one more time and a Raman spectrum was obtained to determine how the OH and OD regions compared. This spectrum was also compared with the Raman spectrum of the sample in the dried state to evaluate how much H₂O was still present. In most cases a third D₂O exchange was not needed but in cases where needed another iteration of replacing old with new D₂O was carried out. OPUS 7.2 software was used to find peak positions and process the spectral data.

Processing of spectra involved normalization, various mathematical operations, and background removal. Background correction was performed using the “rubberband with 64 baseline points option” in OPUS. The method is based on the polynomial fitting of the background. For plotting purposes, the spectra were converted to ASCII format which then allowed the spectral data to be imported to Excel. Details of band intensity calculation and crystallinity determination by Raman are provided in Agarwal 2010. Using OPUS, intensities (peak heights) of the 1380 and 1096 cm⁻¹ bands were calculated by a “one baseline point method” that involved choosing a minimum intensity wavenumber near the peak (e.g., 1440 and 950 cm⁻¹ for 1380 and 1096 cm⁻¹ bands, respectively) and drawing a horizontal line (from that wavenumber) under each peak. Subsequently, the peak heights were measured from this horizontal line. The intensity data were exported to Excel, where Raman band intensity ratios were calculated.

Further regarding crystallinity determination (380 Raman), the sample spectrum was cut in the 250-1850 cm⁻¹ region, baseline corrected (rubber band correction, 64 points), and normalized (min-max) to the 897 cm⁻¹ band intensity of a completely amorphous cellulose sample. From the normalized sample spectrum, the 250-700 cm⁻¹ amorphous cellulose spectrum was subtracted. From the subtracted spectrum of the sample, peak intensities for the 380 and 1096 cm⁻¹ bands were measured using the peak intensity relative to the baseline (between 358 and 396 cm⁻¹) and peak intensity relative to the horizontal baseline (from 944 cm⁻¹) methods, respectively. The intensity ratio I₃₈₀/I₁₀₉₆ was then determined.

Using the previously established correlations (Agarwal 2010 and 2013) that were based on the Bruker RFS-100 and MultiRam instruments, the univariate Raman crystallinities (X) were estimated based on the intensity ratios. Based on RFS-100, the equation below was used for estimating the Raman crystallinity (Agarwal et al. 2010):

X _(MultiRam)=((I ₃₈₀ /I ₁₀₉₆)−0.0286)/0.0065.

However, because MultiRam was used in the present Example, the inter-instrument calibration correction (RFS 100 vs. MultiRam) was carried out using the equation below to get the RFS-100 equivalent crystallinity (XRFS-100):

X _(RFS-100)=(X _(MultiRam)+2.0212)/0.8222.

Additionally, the spectra were corrected for change in the response of the MultiRam optics over time. Using the white light in the sample compartment, the “reference correction” was performed on each sample spectrum.

Further, regarding the accessibility to water measurement from the OH to OD exchange experiments (1380 Raman), the presence of OD groups results in an intensity increase at 1380 cm⁻¹, corresponding to water-accessible CH₂OH groups on cellulose. Water accessibility was calculated by comparing the 1380 cm⁻¹ band intensity increases (ΔI) obtained from samples to the 1380 cm⁻¹ band increases (ΔI) of completely amorphous cellulose and assuming that the CH₂OH groups in the amorphous cellulose were 100% accessible to water. Thus, water accessibility=ΔI₁₃₈₀ cm⁻¹, _(sample)/ΔI₁₃₈₀ cm⁻¹, _(amorphous cellulose))*100.

Crystallinity values obtained via XRD were determined using the Segal method as described in Agarwal, U. P., et al., “Probing Crystallinity of Never-Dried Wood Cellulose with Raman Spectroscopy.” Cellulose 2016, 23 (1), 125-144.

TEM and AFM images were analyzed as follows. In TEM, samples were imaged using a Philips CM-100 TEM operated at 100 kv, spot 3, 200 μm condenser aperture and 70 μm objective aperture. Images were also captured in digital form directly on the microscope using an SIA L3C 4-2 Mpixel CCD camera (Scientific Instruments and Application, Duluth, Ga.). The magnification bars were calibrated using replica grating or asbestos lattice. Image magnifications were 25, 46, and 92K (microscope magnification setting). There is a 1.6× ratio between the original microscope magnification and the camera images. However, since magnification depends on the physical size of the final image, it is much better to refer to the micron bar as a more accurate indicator of size. CNCs widths and lengths were obtained from the images of more than 100 crystals using Image J software (Java-based image analysis program developed at the National Institutes of Health). In such measurements, clumped or aggregated particles in the images were excluded. TEM widths were calculated from the same CNC particles from which lengths were measured.

AFM images were processed using Gwyddion software. The images are first leveled using appropriate functions to ensure a flat background. The background is the reference from which to measure particle height. In order to make a statistical analysis of particle height a mask is created over the image to select the particles, this is done by filtering pixels by height value and slope between pixels. Once the particles are selected, the program calculates and plots the frequency of the occurrence of height values. This results in a bell shape function centered at the mean height (of pixels under the mask) with a spread.

TABLE 3 Comparison of CNCs obtained from Heat Treated Raw Wood and from Wood Pulp. Crystallinity (%) 380 Accessibility to Sample Raman^(a) XRD^(b) water (%) Avg. length (nm) Avg. width (nm) Width range (nm) Sulfur (%) Lignin- NA^(c) 85.5 NA NA  8.2 (TEM) 4.5-11.6 (TEM) 1.6 containing wood-CNCs Lignin-free 64.8 91.5 21 108 (TEM) 10.2 (TEM) 5.0-16.0 (TEM) 2.3 wood-CNCs 7.5 (AFM) 7-8.5 (AFM) Lignin-free 54.5 86.0 26 120 (TEM) 10.7 (TEM)    4-20 (TEM) 1.1 pulp-CNCs ^(a)X_(RFS-100) values. ^(b)Segal method. ^(c)Could not be estimated due to dark coloration of the CNCs.

The results shown in Table 3 show that the lignin-free wood-CNCs are of superior quality as compared to the lignin-free pulp-CNCs. Compared to the lignin-free pulp-CNCs, the lignin-free wood-CNCs exhibit higher crystallinity (an increase of 19%), lower water accessibility, higher surface sulfonation, longer length, smaller width, and narrower width distribution.

Example 2: Crystallinity of, and CNC Extraction from, Raw Wood with Heat Treatment Under Various Conditions

A first set of additional experiments were conducted to crystallize cellulose and isolate CNCs from raw wood. In the experiments, five grams of air-dried, 40 mesh wood (95% solids) in 40 ml of the desired liquid (e.g., pure water) was placed in a teflon-lined Berghof reactor vessel that was sealed, brought to the desired temperature (e.g., 200° C.) over a 75 minute ramp and held at the temperature for a desired period of time (e.g., 90 minutes). Subsequently, the reaction was quenched by placing the vessel in a cold-water bath.

The wood blend was filtered through a sintered glass funnel and washed with reverse osmosis (RO) water until the filtrate was almost colorless. To avoid changes in cellulose crystallinity and hornification, the filter-cake was stored wet in a sealed plastic bag and refrigerated. For lignin containing wood-CNCs, after drying, the filter cake was directly acid hydrolyzed by 64% H₂SO₄ under the conditions described in Example 1. For lignin-free wood-CNCs, the filter-cake was treated at 70° C. over 9 hours with 5 charges of sodium chlorite and acetic acid as per the method of Ahlgren and Goring, Canadian Journal of Chemistry, 49, 1272, (1971). The residual, delignified material was thoroughly washed with RO water. A portion of the material was used to determine crystallinity using 380 Raman as described in Example 1, above, and the rest was used for acid hydrolysis to extract the lignin-free wood-CNCs.

Table 4 shows the CNC yields and crystallinity values for the samples using different wood types, different liquids (or no liquid), different temperatures for the heat treatment, and different time periods for the heat treatment. CNC yields and crystallinity values were obtained as described in Example 1, above.

In a second set of experiments, raw wood was first thermomechanically pulped prior to crystallizing cellulose via a heat treatment and extraction of CNCs. In these experiments, softwood chips were thermomechanically pulped in a pressurized disc refiner. Chips (4.5 kg) were steamed at 167° C. for 10 minutes, at which time they were passed through Sprout-Waldon 12-1CP pressurized disk fitted with D2B505 plates at a gap of 0.1 mm consuming 275 W-hr of energy during the 7 minutes it took to refine the batch of chips. This pulp was then air dried (MDF5).

The pulp was then subjected to a heat treatment under different conditions. For each, 52.5 g of air-dry pulp was mixed with 950 mL water and placed in a stirred, horizontal Parr reactor. The reactor was heated to the desired temperature (taking 60-90 min) then held at that temperature for the desired time before quenching the reaction by placing the reactor in a bucket of cold water. The pulps were filtered, washed and air dried to determine pretreatment yield.

Extraction of CNCs was as follows, for lignin containing CNCs, the pulp was directly acid hydrolyzed by 64% H₂SO₄ under the conditions described in Example 1. For lignin-free CNCs, the pulp was treated at 70° C. over 9 hours with 5 charges of sodium chlorite and acetic acid as described above. The residual, delignified material was thoroughly washed with RO water. A portion of the material was used to determine crystallinity using 380 Raman as described in Example 1, above, and the rest was air dried and used for acid hydrolysis to extract the lignin-free CNCs.

The results are shown in Table 4, below.

TABLE 4 Cellulose crystallization and CNC extraction from raw wood. Treatment time Crystallinity^(b) Wood Method/Reactor Liquid Temperature (° C.) (min) CNC Yield^(d) (%) (380 Raman) Loblolly pine None NA^(a) NA NA 2 46.6 Loblolly pine Delignified H₂O 70 (used during 300 3 48.0 delignification) Loblolly pine Berghof^(c) H₂O 170 90 11 54.4 Aspen Delignified H₂O 70 (used during 540 ND^(c) 45.3 delignification) Poplar Berghof H₂O 100 90 ND ND Poplar Berghof None 135 385 ND 48.8 Poplar Berghof H₂O 135 90 ND 50.1 Poplar Berghof H₂O 170 720 20 55.3 Poplar Berghof H₂O 200 90 17 65.7 Poplar Berghof 4M CaCl₂ in 200 90 ND 51.7 H₂O Poplar Berghof 4M LiCl in H₂O 200 90 ND 55.3 Poplar Berghof 4M ZnCl₂ in 200 90 ND ND H₂O Poplar Berghof 0.1M K 200 90 ND ND phosphate buffer in H₂O Poplar Berghof 1M K 200 90 ND ND phosphate buffer in H₂O Yreka^(f) Berghof None 170 390 ND ND Yreka Berghof H₂O 170 90 ND ND Yreka MDF5 Disc refiner H₂O 167^(g) 10-20^(g) 8.0 57.2 Yreka Disc refiner, Parr^(h) H₂O 167^(g), 200^(h) 10-20^(g), 90^(h) 8.6 65.7 MDF5 Yreka Disc refiner, Parr H₂O 167^(g), 225^(h) 10-20^(g), 30^(h) 11.5 65.0 MDF5 ^(a)Not applicable. ^(b)Except for Loblolly pine wood crystallinity in first row, all crystallinity data is on processed raw wood. ^(c)Reactor used for the heat treatment. ^(d)CNC yield is determined from lignin-containing wood-CNCs as described in Example 1. For untreated loblolly pine, 2% yield shown contained 31% lignin and it was not clear that any CNCs were present. ^(e)ND indicates that the measurement was not conducted. ^(f)Yreka is a combination of softwoods including ponderosa pine, Douglas fir, and white fir. ^(g)In disc refiner (10-20 min) at 167° C. then in Parr (90 or 30 mm) at 200 or 225° C. ^(h)Reactor used for the heat treatment.

The word “illustrative” is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “illustrative” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Further, for the purposes of this disclosure and unless otherwise specified, “a” or “an” means “one or more”.

The foregoing description of illustrative embodiments of the invention has been presented for purposes of illustration and of description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention. The embodiments were chosen and described in order to explain the principles of the invention and as practical applications of the invention to enable one skilled in the art to utilize the invention in various embodiments and with various modifications as suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents. 

What is claimed is:
 1. A method for forming crystalline cellulose in raw wood, the method comprising subjecting raw wood comprising cellulose, lignin and hemicellulose to a heat treatment at a crystallization temperature in the range of from about 150° C. to about 250° C. for a period of time sufficient to induce crystallization of cellulose in the raw wood, wherein the crystallinity of the processed raw wood as measured after delignification using 380 Raman is at least 5% greater than the crystallinity of the raw wood as measured after delignification and prior to processing using 380 Raman.
 2. The method of claim 1, wherein the crystallinity of the processed raw wood as measured after delignification using 380 Raman is at least 12% greater than the crystallinity of the raw wood as measured after delignification and prior to processing using 380 Raman.
 3. The method of claim 1, wherein the period of time is at least 30 minutes.
 4. The method of claim 1, wherein the crystallization temperature is in the range of from about 170° C. to about 230° C.
 5. The method of claim 1, wherein the raw wood is provided as a wood blend comprising the raw wood, water and optionally, a salt, a buffer, or a combination thereof.
 6. The method of claim 5, wherein the wood blend consists essentially of the raw wood, water and optionally, the salt, the buffer, or the combination thereof.
 7. The method of claim 1, wherein the processed raw wood exhibits an accessibility to water of less than 26% as measured from the processed raw wood after delignification using 1380 Raman.
 8. The method of claim 1, wherein the processed raw wood exhibits an accessibility to water of less than 20% as measured from the processed raw wood after delignification using 1380 Raman.
 9. The method of claim 1, further comprising removing lignin from the processed raw wood via a delignification procedure.
 10. The method of claim 1, wherein the heat treatment does not convert the raw wood to pulped wood.
 11. A method of producing cellulose nanocrystals (CNCs) from raw wood, the method comprising: subjecting raw wood comprising cellulose, lignin and hemicellulose to a heat treatment at a crystallization temperature in the range of from about 150° C. to about 250° C. for a period of time sufficient to induce crystallization of cellulose in the raw wood, wherein the crystallinity of the processed raw wood as measured after delignification using 380 Raman is at least 5% greater than the crystallinity of the raw wood as measured after delignification and prior to processing using 380 Raman; and extracting CNCs from the processed raw wood via an acid hydrolysis procedure.
 12. The method of claim 11, wherein the extracted CNCs comprise lignin.
 13. The method of claim 12, wherein the Klason lignin content of the CNCs is about 20% or greater.
 14. The method of claim 11, further comprising removing lignin from the processed raw wood via a delignification procedure prior to extraction of CNCs.
 15. The method of claim 11, wherein the CNCs are characterized by an average length in the range of from about 50 nm to about 350 nm as measured using transmission electron microscopy (TEM).
 16. The method of claim 11, wherein the CNCs are characterized by an average width in the range of from about 4 nm to about 20 nm as measured using TEM.
 17. The method of claim 16, wherein the width distribution of the CNCs is less than about 2 nm as measured using atomic force microscopy (AFM).
 18. The method of claim 11, wherein the heat treatment does not convert the raw wood to pulped wood.
 19. Cellulose nanocrystals comprising lignin and having a Klason lignin content of about 20% or greater. 